Modeling ASD-linked mutations in 3D human brain organoids Neuropsychiatric diseases are very complex, and we still have a limited understanding of the abnormalities associated with genetic mutation in these pathologies. This is in part complicated by the lack of single, ideal experimental models for these overtly ?human? diseases, and the need to investigate abnormalities in diverse genetic backgrounds. Rodent models have proven valuable to highlight phenotypic abnormalities associated with autism spectrum disorder (ASD)-linked mutations. However, rodents differ in the development, architecture and function of their brain compared to humans making discoveries difficult to relate to patients. We share the vision of this consortium that integrated investigation of multiple experimental models, including models of the human brain, is needed to progress understanding of ASD. Studies using human brain tissue are complicated by practical and ethical concerns of tissue availability, expansion and manipulation. However, recent progress has enabled the development of cellular models of the human developing brain via the generation of 3D brain organoids, which we propose can complement rodent and non-human primate systems to model basic aspects of human pathology. Although reductionist in nature, 3D brain organoids are amenable to high-throughput genetic engineering and can provide a valuable platform to link mutations in disease-associated genes with specific abnormalities in human neurons and circuits, as well as help identify molecular targets. Here, we will use a protocol that we recently established to generate long-term cultures of human brain organoids engineered to carry the same mutations in the SHANK3 and MECP2 genes investigated in rodents and marmosets by the other members of the consortium. We will pioneer extensive molecular, morphological and electrophysiological analysis of mutant and control organoids to understand whether these mutations induce defects in human neurons and networks similar to those observed in mice, and to generate a transcriptional map of molecular changes that informs mechanistic understanding. In addition, we will optimize our recent Method for Analyzing RNA following Intracellular Sorting (MARIS) to molecularly profile specific subclasses of cortical neurons from rodent and marmoset brain and brain organoids. This will provide the first inter-species comparison of disease-relevant mutant and control neurons in three model systems to highlight molecular abnormalities and pinpoint cell type-specific defects.